Procaine penicillin and therapeutic compositions



Patented July 18, 1950 PROCAINE PENICILLIN AND THERAPEUTIC COMPOSITIONS Harley W. Rhodehamel, Jr., Indianapolis, Ind.,

assignor to Eli Lilly and Company, Indianapolis, Ind., a corporation of Indiana No Drawing. Application September 15, 1947,

Serial No. 774,183

Claims. i

This invention relates to relatively insoluble salts of penicillin, particularly procaine penicillin, to the preparation of such salts, and to compositions embodying them.

In the past, penicillin has been prepared and used in the form of its simple salts, usually the sodium or potassium salt. Such salts are highly soluble in water and in the body fluids and tend to decompose in their presence. When such salts are injected, as in water solution, the penicillin not only tends to decompose, but is rapidly taken up in the body fluids and is soon eliminated in the urine; so that the penicillin concentration in the blood remains at an effective level for but a short period of time. Because of this, when the penicillin is administered in aqueous solution, it has been necessary to give frequent injections, usually every three or four hours. Penicillin therapy requires the maintenance of effective blood concentrations over prolonged periods, usually for several days, and the necessity for frequent iniections is extremely burdensome both to the doctor and to the patient. Many attempts have been made to eliminate thenecessity for these frequent injections and to obtain some means of producing an efiective blood concentration of pencillin over a prolonged period, desirably at least 24 hours; without the necessity for such frequent injections.

One method which has been used for this purpose is by injecting the penicillin in suspension in a mixture of oil and beeswax; and while with proper concentrations and especially with suiilcient beeswax, this has been somewhat successful in producing prolonged effective concentrations of penicillin, its use is disagreeable to the patient, it may cause fibrosis, and it leaves a residual mass of beeswax which may not be absorbed by the body for several months and which in some cases requires surgical removal.

By my invention, I provide a salt of penicillin which is relatively insoluble in water and in body fluids and is non-toxic, which can be administered without discomfort to the patient and to leave no residue, and which will readily provide a supply in the body from which the penicillin will slowly absorb to maintain effective concentrations of penicillin in the blood stream over prolonged periods of time. Whereas with the known salts of penicillin maintenance of effective concentrations of penicillin requires eight or more aqueous injections per day, with my salt effective concentrations may be maintained with but a single aqueous injection a day.

I have found that procaine forms a salt with penicillin which is relatively insoluble in water 2 and in the body fluids, and that infrequent administrations of this salt, usually not oftener than once each day, will readily maintain effective concentrations of penicillin in the blood over prolonged periods.

Small quantities of procaine have previously been administered in conjunction with penicillin therapy, in part to obtain the anesthetic effect of the procaine, and in part because the procaine was found in many cases to minimize or overcome he allergy which some persons have toward peniillin. My new procaine-penicillin salt not only permits the maintenance of effective blood concentrations of penicillin over prolonged periods, but also provides the procaine for its anesthetic eifect and its anti-allergy effect.

The pencillin embodied in the'salt may be a mixture of one or more of the several specific penicillins, as obtained in the usual production of penicillin, or may be a penicillin which consists mainly or wholly of one of these specific penicillins. Preferably, I use a penicillin which is rich in penicillin G (benzyl penicillin), in part for the excellent therapeutic qualities of penicillin G but also because I find it especially useful in forming salts with desirable physical properties. The penicillin used may be either an amorphous penicillin or preferably a crystalline penicillin; and in preparing the salt, I start with either a dry penicillin or a purified solution containing the penicillin, and such solution may be One obtained directly, as by extraction, from the broth in which the penicillin is produced.

The new procaine penicillin salt may be administered in any desired way. The usually preferred way is by intra-muscular injection of a suspension of the procaine penicillin in water or other aqueous menstruum, i. e. aqueous suspending medium, or in any of the commonly used oily menstruums, i. e. oily suspending media. when it is to be administered in water suspension, the suspension is desirably a freshly prepared one, because of thetendency of the penicillin to decompose in the presence of water. For such administration, the solid procaine penicillin in an amount to provide a unit dosage, desirably about 200,000 to 300,000 international penicillin units, is put up in a sealed ampoule in combination with an ampoule of a suitable quantity of aqueous menstruum, so that the doctor may combine the two shortly before administration. In such a combination package, the procaine penicillin salt is contained in its ampoule in finely divided state, preferably less than 40' mesh size and most desirably about mesh slze. The amount of aqueous menstruum supplied in the combined vial is desirably small, but is sufficient to form a suspension of a consistency suitable for injection. With 200,000 to 300,000 units of procaine penicillin having a potency of 1,000 units per mg., I desirably use about 1 ml. of aqueous menstruum. My procaine penicillin salt lends itself to this advantageous method of administration, for it forms crystals which may be sufllciently finely divided to form the suitable suspension, and such finely divided crystalline material lends itself to convenient packaging in ampoules and when so packaged remains stable over long periods and can be distributed by usual methods and through usual channels.

To provide a composition which may be administered directly as supplied, my penicillin salt is also of advantage. line material may be suspended in any of the commonly used injectable oily menstruums, and

when so suspended in the absence of water, the

suspension may be distributed through the usual channels in the usual way and will remain stable and available for immediate use .without preliminary mixing.

My procaine-penicillin salt may be prepared by simply bringing the procaine and the penicillin together, desirably with the procaine in excess, in solution in a suitable reaction solvent. The procaine penicillin is relatively insoluble both in water and in convenient organic solvents so that it precipitates from the reaction mixture, usually quite promptly as a solid, and is conveniently recovered by filtration.

For preparation in aqueous solvent, the penicillin is conveniently and desirably added in the form of any of its usual soluble salts, such as the ammonium, potassium, sodium, or calcium salts. The procaine may likewise be present in the form of its soluble salt, such as its hydrohalide, sulfate, nitrate, formate. or the like.

The aqueous reaction mixture may be prepared in any convenient way; one of the reactants, conveniently the penicillin, may be provided in solution in sufiicient water for the reaction mixture, and the other reactant added thereto, or preferably, water solutions of each of the reactants are combined. When the penicillin and the procaine are brought together in solution in the aqueous reaction mixture, a precipitation promptly occurs,

of the desired procaine-penicillin salt. To increase recovery, the reaction mixture is desirably allowed to stand in the cold. The procaine penicillin is recovered, as b filtration, and is then preferably washed first with water and then with a low-boiling water miscible solvent, such as acetone, and then dried under vacuum.

For preparation in an organic solvent, the penicillin is desirably supplied in acid form, and the The finely divided crystalprocaine desirably in base form. As in the aqueous preparation, the two reactants and the solvent may be brought together in any desired order. When the penicillin, in acid form, and the procaine, in base form, are brought together in organic solution, they react to form the desired procaine-penicillin salt, which precipitates. The precipitate is recovered by filtration, and is desirably washed with an additional quantity of organic solvent and dried.

For preparation of the procaine penicillin in organic solution, I preferably use as the reaction solvent an alkyl acetate 01' from about 4 to 9 carbon atoms, and most desirably use amyl acetate. Other solvents, such as ether or chloroform, in which the two reactants are soluble and the procaine-penicillin is relatively insoluble, may also be used; but I find that with such other solvents the procaine-penicillin sometimes precipitates as an oil instead of in the more desirable, solid crystalline state, and that sometimes the occurrence of precipitation is delayed. In the preparation of procaine penicillin, both in water solution and in organic solvent solution, the penicillin must of course be present in a, concentration greater than the solubility of procaine penicillin in the solvent (about 0.7% in water at 28 C.), and for efllcient recoverythe penicillin should be present in a substantially higher concentration, desirably close to its maximum solubility. In addition, the penicillin used should be of fairly high purity, desirably of at least purity, for the co-presence of excessive impurities may interfere with the formation of the procaine penicillin or with its recovery in crystalline form. For example, with dry sodium or potassium penicillin G, which when pure have potencies of about 1,667 units and 1,595 units per mg. respectively, I have found it suitable to use a penicillin having a potency of about 1200 units per mg., but I prefer to use a penicillin of substantially higher potency.

With both the aqueous and the organic-solution reactions, the penicillin is desirably brought to the reaction mixture in solution, and the penicillin-containing solution may be-one which is obtained directly by extractive procedure from the broth in which the penicillin is produced. The penicillin-containing solution may for example be the first organic extract of the broth, or may be the first aqueous extract thereof, derived by extraction of that organic extract, or may be a subsequent extract, either aqueous or organic. When the penicillin is obtained in such an extract from the broth, it is desirably decolorized in organic solution before use by any suitable method.

The procaine penicillin obtained by the procedures set forth above is usually obtained in the form of crystals, although sometimes, especially when the reaction solvent is other than water or amyl acetate, the procaine penicillin may precipitate as an oil which solidifies on standing.

For use in injectable suspensions I employ crystalline procaine penicillin in finely divided state, preferably about 40 to about mesh size. The crystal size obained in the preparation of my procaine penicillin may be modified by suitably controlling the conditions under which the reaction is carried out, and crystal size is reduced by carrying out the reaction at reduced temperature and with vigorous stirring. In any event, the material if not obtained in a sufficient state of division readily lends itself to further reduction by grinding, as through a screen of suitable mesh.

The following are examples of the preparation of procaine penicillin.

Example 1.To a solution of 75 grams (containing about 120,000,000 units) of crystalline sodium penicillin G in 450 ml. of distilled water is added a solution of 75 grams of procaine hydrochlorlde in 200 ml. of distilled water. A precipitation occurs almost immediately, of the desired procaine penicillin G. The mixture then is cooled as by placing it in the ice box for about 2 hours. It is then filtered, and the procaine penicillin is washed with 500 ml. of distilled water and then with 250 ml. of acetone, and the washed material is then dried for about 12 hours under vacuum. The dried procaine penicillin G is obtained in a yield of about 112 grams, with a potency of about 1000 units per mg.; or a total recovery of about 112,000,000 units, which represents a conversion and recovery penicillin of about 93% of the potency contained in the original sodium penicillin. The penicillin remaining in the mother liquor may be recovered by known methods to give a material balance of substantially 100%.

Example 2.Example 1 is repeated save that instead of using crystalline sodium penicillin G, I use crystalline potassium penicillin G. The procaine penicillin is obtained in about the same potency and in about the same yield.

Example 3.--A penicillin-producing mold is grown in the usual way in an aqueous nutrient medium to obtain a penicillin content in the broth of about 300 units per ml. The broth is separated from the mold growth by filtration, is adjusted to about pH 2 to 3, and is extracted with amyl acetate in an amount about one-fifth to onefourth the volume of the filtered broth. This first amyl acetate extract is decolorized, by stirring with a small amount of decolorizing carbon for about 15 minutes, and is then separated from the carbon by filtration. The decolorized first organic extract is adjusted to about pH 6.5 to '7 and extracted with a dilute aqueous sodium hydroxide solution to obtain a first aqueous extract, containing sodium penicillin in a concentration of about 10,000 to 12,000 units per m1.

To 1,000 ml. of this first aqueous extract is added a solution of about six grams of procaine hydrochloride in 15 ml. of distilled water, with stirring. A precipitate occurs almost immediately, of the desired procaine penicillin. The mixture is then placed in the refrigerator for about 2 hours and is then filtered. The solid procaine penicillin so recovered is washed with about 40 m1. of distilled water and then with about 20 ml. of acetone and then dried for about 4 hours under vacuum. The dried crystalline procaine penicillin has a potency of about 900 units per mg.

Example 4.-Three grams of dry amorphous sodium penicillin (having a potency of about 1,300 units per mg.) is dissolved in about 30 ml. of water. To this solution is added a solution of about three grams of procaine hydrochloride in ml. of water, with stirring. An almost immediate precipitation occurs, of crystalline material. The reaction mixture is placed in the refrigerator over night, and is then filtered to recover the crystalline material, which is the desired procaine penicillin. This is desirably washed, first with a small amount of water and then with a small amount of acetone, and then dried under vacuum. In a preparation according to this example, the yield was about 2.9 grams of crystalline procaine penicillin, having a potency of about 900 units per mg., representing a conversion recovery in procaine penicillin of about 70% of the original potency of the amorphous sodium penicillin.

Example 5.-To 1000 ml. of the decolorized first organic extract obtained as set forth in Example 3 is added with stirring about 2 grams of procaine (as the base) which for convenient handling may be dissolved in about ml. of amyl acetate. A precipitate occurs almost immediately. The stirring may be continued for about a quarter to half an hour. To insure maximum recovery, the reaction mixture is placed in the refrigerator for about two hours. The precipitate is the desired procaine penicillin, which is recovered by filtration and is desirably washed with a small amount of amyl acetate and dried under, vacuum.

Example 6.Ten grams of sodium penicillin G are dissolved in 1000 ml. of water and a sufficient amount (about 14 ml.) of a l-molar solution of in the procaine mixture sulfuric acid is added to obtain about pH 2. .The desirably cooled and stirred durin this add tion. The resulting mixture is extracted with 1,500 ml. of amyl acetate at a temperature of about 0 C. The amyl acetate is allowed to form a separate layer, which is recovered.

To this amyl acetate solution, which contains the penicillin, are added about 10 grams of procaine (as the base), conveniently in solution in about ml. of amyl acetate, and the mixture is stirred for about half an hour. A precipitate occurs almost immediately, and to insure complete recovery the reaction mixture is cooled in the refrigerator for about two hours. The precipitate is the desired procaine penicillin, which is recovered by filtration and is desirably washed with amyl acetate and dried.

Example 7.-Examples 3, 4, and 6 may be repeated save that instead of using sodium penicillin, I may use potassium penicillin.

Example 8.Any of Examples 1 to 4 and 6 may be repeated save that instead of using sodium or potassium penicillin, I may use another inorganic salt, or an organic soluble salt of penicillin, such as the calcium salt, the ammonium salt, the N- ethyl piperidine salt, etc.

Example 9.Examples 5 and 6 may be repeated save that instead of using amyl acetate as the re action solvent I may use other organic solvents in which the penicillin and the procaine are soluble and in which the procaine penicillin is relatively insoluble, or I may use a mixture of organic solvents. Other organic solvents which may be used are other alkyl acetates of from 4 to 9 car- I and hexyl acetate.

bon atoms, such as butyl acetate, isoamyl acetate, I may also use such solvents as ether and chloroform, but with these the procaine penicillin sometimes precipitates as an oil instead of in crystalline form.

The following are examples of injectable suspensions oi procaine penicillin suitable for penicillin therapy and which give prolonged effective blood concentrations.

Example 10.--Crystal1ine procaine penicillin, such as that produced in Example 1 above, and having a potency of about 1,000 units per mg., and in a finely divided state (conveniently about mesh), is put up in vials each containing about 200 mg., or a potency of about 200,000 units. A second vial is combined with each of such procaine-penicillin containing vials and contains about 1 ml. of distilled sterile water. Shortly before administration--which may be several hours before administration-the water-containing vial is emptied into the procaine penicillin containing vial, and the mixture is shaken to produce a suspension. The suspension is taken up in a hypodermic syringe, and is administered by intramuscular injection.

In a typical case. in a patient receiving an intramuscular injection of an aqueous procainepenicillin suspension prepared as set forth in this example and containing only 200,000 units of penicillin activity, blood levels of penicillin concentration were maintained as follows:

At the end of the 24 hour test period, the blood concentration still remained at an effective level of 0.06 units per ml.

Example 11.-Crystalline procaine penicillin G, having a potency of about 1,000 units per mg. and in a state of fine division (conveniently about 40 mesh) is suspended in dry sterile vegetable oil, such as cottonseed oil, peanut oil, and sesame oil, in an amount suflicient to provide about 300,000 units per ml. of suspension. This is packaged in suitable vials, conveniently containing about 10 ml. Such vials may be packaged and shipped as such, preferably under conditions which prevent a rise of temperature above about 50 0.; and provide a unitary product ready to be injected intramuscularly to provide a supply of penicillin which is slowly absorbed and which readily maintains efi'ective penicillin blood concentrations for periods in excess of 24 hours.

In a typical case, in a patient receiving an intramuscular injection of one ml. of a suspension of crystalline procaine-penicillin in cottonseed oil, with the suspension containing about 300,000 units of penicillin per ml., and with the procaine penicillin in about 40-mesh size, blood levels of penicillin were maintained as follows:

Penicillin blood-concentration f i gg f' Hours 0.406 unit per ml 1 0.248 unit per ml 0.124 unit per ml 0.124 unit per m1 Example 12.Instead of the simple water or oily menstruums of Examples 10 and 1'. I may use mixed menstruums, or menstruums which themselves tend to prolong the effective blood concentrations of penicillin. One such men- .cottonseed oil, with procaine penicillin G present in an amount to give about 300,000 units per ml.;

and the emulsionyvas stabilized with about 0.04%

of a water-soluble emulsifying agent and about 0.04% of an oil-soluble emulsifying agent, and

025% phenol was added as a preservative. In a typical case, in a patient receiving 1 m1. of this suspension, blood levels of penicillin were maintained as follows:

I claim as my invention:

1. A solid procaine salt of penicillin.

2. A solid procaine salt of penicillin G.

3. A therapeutic composition comprising a liquid suspending medium, and finely divided solid procaine penicillin in an amount greater than will dissolve to saturate the suspending medium;

4. A therapeutic composition comprising a liquid injectable suspending medium and solid procaine penicillin having a particle size less than about 40 mesh, said procaine penicillin being present in an amount greater than will dissolve to saturate the suspending medium.

5. A composition according to claim 4 in which the procaine penicillin is procaine penicillin G.

6. An injectable therapeutic composition comprising a liquid injectable suspending medium and solid procaine penicillin having a particle size less than about 40 mesh, said procaine penicillin being present in an amount greater than about 200,000 units per milliliter.

7. A composition according to claim 6 in which the suspending medium is an aqueous suspending medium.

8. A composition according to claim 7 in which the procaine penicillin is procaine penicillin G.

9. A composition according to claim 6 in which the suspending medium is a vegetable oil.

10. A composition according to claim 9 in which the procaine penicillin is procaine penicillin G.

HARLEY W. RHODEHAMEL, JR.

REFERENCES CITED The following references are of record in the file of this patent:

Proc. Soc. Exptl. Biol. and Med., June. 1942, pp. 285 to 288.

Science, Sept. 1, 1944, pp. 196 to 198.

Science, Nov. 3, 1944, pp. 412 and 413.

Lancet, Nov. 11, 1944, g. 623-167-65P.

Proceeding of Staff Meeting of Mayo Clinic, Feb. 7. 1945, pp. 40, 41.

Journal American Med. Assoc, July 21, 1945. p. 910.

British Reports 234, pp. 1-5, Feb. 12, 1946.

J. Amer. Phar. Assoc. Pract. Pharm. Ed., Am 1946, p. 363. 

1. A SOLID PROCAINE SALT OF PENICILLIN.
 3. A THERAPEUTIC COMPOSITION COMPRISING A LIQUID SUSPENDING MEDIUM, AND FINELY DIVIDED SOLID PROCAINE PENICILLIN IN AN AMOUNT GREATER THAN WILL DISSOLVE TO SATURATE THE SUSPENDING MEDIUM. 